Methods and compositions for screening cloned proteins

ABSTRACT

The present invention provides a metal charged iminodiacetic acid (IDA) cellulose for detecting a test sample having a histidine tag. The present invention also provides methods for determining cloned protein expression and function. Additionally, the present invention includes a method for the handling of denatured proteins with subsequent renaturation in situ (parenthetically after binding to metal charged IDA cellulose). A wide range of applications are contemplated for the metal charged IDA cellulose including two-dimensional high throughput screening of proteins.

This invention was made with government support under Grant No. ES-04068awarded by the National Institutes of Health. The Government has certainrights in the invention.

BACKGROUND OF THE INVENTION

Screening cloned prokaryotic or eukaryotic hosts for protein expressionand function is of great importance in the field of biotechnology.However, this technology requires evaluating each individual clone forprotein expression and function. Often it is necessary to examine largenumbers of cloned hosts to determine which expression system is mostefficient or which protein has the characteristic function that is mostdesirable.

Mutagenesis is a powerful method for evaluating protein expression andfunction. Mutagenesis involves modifying one or more bases in anucleotide sequence to express the protein of interest. However, becausethere are no reliable methods to predict the effect of modifiednucleotide sequences, large numbers of point mutants and mutant proteinsneed to be individually evaluated.

In the past, screening individual mutants for protein expression andfunction was extremely time consuming and very labor intensive becauseindividual samples needed to be evaluated. It was also very difficult todetermine which point mutant expressed the protein of interest.

Proliferating cell nuclear antigen (PCNA) exemplifies this difficulty.Previously, the evaluation of PCNA point mutants was performed bytesting individual clones for their effects in vitro on purified DNApolymerase δ (pol δ) using conventional protein identification andpurification techniques of mutant PCNA molecules. This approach whileeffective, is very labor intensive. (See, for example, Refs. 9-11).

The isolation and purification of cloned proteins has been greatlyfacilitated by the use of affinity tags. A widely used affinity tag is ahistidine (his) tag. These tags typically contain a string of four toten consecutive histidine residues genetically engineered to be ateither the amino- or carboxyl-terminus of recombinant proteins. Becausehis-tagged proteins bind to chelated metals attached to solid supports,they can easily be isolated by column chromatography with chelatedmetals attached to resins or beads as the solid support. Suchchromatographic methods are referred to as Immobilized Metal IonAffinity Chromatography (IMAC).

The use of IMAC for isolating proteins was first disclosed in Porath etal., (Ref. 31), wherein a resin was derivatized with iminodiacetic acid(IDA) and metal ions were chelated to the IDA-derivatized resin forimmobilizing proteins. Columns with nickel-agarose or metal containingresin are typically used for isolating his-tagged proteins. Thehis-tagged protein binds to the matrix by interacting with the metalions and is then eluted with a solution of imidazole, competing metalions or salt. Unfortunately, the use of columns for detecting his-taggedproteins is very labor intensive and not amenable to high throughputscreening.

An alternate method for identifying his-tagged proteins is disclosed inthe QIAGEN product guide 1997. The QIAGEN method is a two dimensionalmethod for detecting his-tagged proteins that uses Ni-nitrilotriaceticacid (Ni-NTA) attached to a column matrix to bind his-tagged proteins.

Similarly, a method for identifying his-tagged proteins on micro titterplates is disclosed in Paborsky et al., (Ref. 29). This method utilizesmaleic anhydride-activated polystyrene microtiter plates coupled withN,N-bis[carboxymethyl]lysine, a derivative of nitrilotriacetic acid(NTA), to immobilized histidine-containing proteins. Paborsky alsodiscloses a method for quantifying expression levels of the immobilizedprotein. These methods, although useful, are very labor intensive andnot amenable to high throughput screening.

Based on the foregoing, there is still a great need for alternatemethods and compositions for screening large numbers of cloned mutantsfor both protein expression and function. Methods and compositions thatallow two-dimensional high throughput screening would be of particularvalue for designing proteins for pharmaceutical and industrial uses.

SUMMARY OF THE INVENTION

These and other objectives are achieved by the present invention, whichin one embodiment provides a modified cellulose for detecting a proteinof interest, comprising metal charged iminodiacetic acid cellulose.

In another embodiment, the present invention provides a process forpreparing modified cellulose for his-tag protein binding, comprisingreacting cellulose epoxide with iminodiacetic acid to form iminodiaceticacid-cellulose; and incubating the iminodiacetic acid-cellulose with ametal salt thereby preparing a metal charged iminodiacetic acidcellulose for binding his-tagged proteins.

In yet another embodiment, the present invention provides a method forscreening a test sample to determine if the sample is his-tagged,comprising contacting metal charged iminodiacetic acid cellulose withthe test sample; washing the iminodiacetic acid-cellulose; and detectingthe his-tagged sample that remains immobilized on the metal chargediminodiacetic acid cellulose.

In one embodiment, the invention includes a method for determiningprotein expression, comprising introducing into cells a vectorcomprising a nucleic acid that encodes a protein of interest having apolyhistidine region; growing the cells that have the vector; preparinga replica of the cells that have the vector on a membrane support;expressing the protein of interest in the cells; lysing the cells on themembrane support in situ to release the protein of interest;transferring the protein of interest to a metal charged iminodiaceticacid cellulose; washing the metal charged iminodiacetic acid cellulose;and detecting the protein of interest that is immobilized on the metalcharged iminodiacetic acid cellulose.

In another embodiment, the present invention includes a method forrenaturing proteins, comprising denaturing a protein having apolyhistidine region; contacting the protein with metal chargediminodiacetic acid cellulose to transfer and bind the protein to thecellulose under denaturing conditions; renaturing the protein; andrecovering or detecting the renatured protein.

A significant advantage of the metal charged iminodiacetic acidcellulose is that it allows for easy screening of a large number ofproteins following mutagenesis. Accordingly, one skilled in the art canrapidly ascertain which mutants have desired functional activity orbinding capacity.

The invention provides, for the first time, a two-dimensional screeningsystem that is readily amenable to high throughput screening of clonedproteins. By maintaining a two-dimensional format, the present inventionenables the processing of large numbers of samples concurrently. This isnot possible with the three dimensional columns and gels available inthe prior art.

These and other advantages of the present invention will be appreciatedfrom the detailed description and examples set forth herein. Thedetailed description and examples enhance the understanding of theinvention, but are not intended to limit the scope of the invention.

DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic diagram showing the preparation and composition ofthe metal charged IDA cellulose of the present invention.

FIG. 2 shows SDS-PAGE and immunoblot analysis of bacterial expressionand purification of unmodified and NH₂-terminally his-tagged PCNA; (A)gel was stained with Coomassie blue; (B) immunoblot probed with mAbPC10; and (C) immunoblot probed with affinity purified rabbit polyclonalanti-Drosophila PCNA antibody.

FIG. 3 shows binding of bacterial lysates containing either his-tagged(spots labeled 1) or unmodified (spots labeled 2) human PCNA; (A)binding of his-tagged PCNA to Ni²⁺-IDA-paper; (B) binding of PCNA toIDA-paper (not metal charged); (C) binding of PCNA to unmodified 3MMpaper; (D) binding of PCNA to Ni²⁺-charged IDA-paper when incubated for5 min in 600 mM imidazole after lysate application; and (E) binding ofPCNA to nitrocellulose.

FIGS. 4(A), (B) and (C) show in situ reactivity of his-tagged humanPCNA, purified calf thymus pol δ and exogenous DNA template-primer.Lysates applied to spots labeled 1 contained his-tagged human PCNA andlysates applied to spots labeled 2 contained unmodified human PCNA; (D)the nucleotide sequence of exogenous DNA template primers used in pol δreaction; and (E) shows in situ reactivity of purified calf thymus pol δand exogenous DNA template—primer with human PCNA but not withDrosophila PCNA.

FIG. 5 shows growth of bacterial colonies, specific immobilization ofhistagged human PCNA on Ni²⁺-IDA paper and in situ reactivity ofpurified calf thymus pol δ; (A) shows bacterial colonies on a celluloseacetate membrane; (B) shows Ni²⁺-IDA paper after macromolecular transfersubjected to immunoblot-type analysis with mAb PC10; (C) shows Ni²⁺-IDApaper after macromolecular transfer subjected to DNA polymerase analysislacking pol δ; (D) shows Ni²⁺-IDA paper after macromolecular transfersubjected to DNA polymerase analysis; and (E) shows Ni²⁺-IDA paper aftertransfer and washing subjected to Coomassie blue staining.

FIG. 6(A) shows Coomassie blue staining of bacterial lysates applieddirectly to Ni²⁺-IDA-paper. The lysate applied in segment 1 containedhis-tagged human PCNA. The lysate applied in segment 2 contained anequal amount of unmodified human PCNA; and (B) is a graphic illustrationof densitometric quantifications of his-tagged protein binding toNi²⁺-IDA paper.

FIG. 7(A) shows Ni-IDA paper screened by immunoblot-type assay with mAbPC10; (B) shows Ni-IDA paper screened by in situ polymerase assay usinga lesion-containing DNA template-primer; (C) SDS-PAGE/Coomassie blueanalysis of highly purified recombinant PCNA: lane 1, unmodifiedwild-type human PCNA; lane 2, NH₂-terminally his-tagged wild-type humanPCNA; lane 3, NH₂-terminally his-tagged E⁸⁵>K mutant human PCNA; (D)lesion-containing DNA template-primer used for the screen; (E) solutionassays of pol δ in the presence and absence of different PCNA molecules:lane 1, no PCNA; lane 2, unmodified wild-type human PCNA; lane 3,NH₂-terminally his-tagged wild-type human PCNA; lane 4, NH₂-terminallyhis-tagged E⁸⁵>K mutant human PCNA; and (F) location of the pointmutation found (E⁸⁵>K) superimposed on the crystal structure of the PCNAtrimer.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a novel modified cellulose for detectinga protein of interest. The modified cellulose comprises metal chargediminodiacetic acid (IDA) cellulose. The metal charged IDA cellulose ofthe present invention is a two-dimensional matrix that allows for highthroughput screening. Examples of two dimensional matrices includepaper, membranes, filters, and the like.

For purposes of the present invention, two dimensional metal charged IDAcellulose is derived generally from a sheet or membrane of cellulosicfibers. As used herein, cellulose includes any convenient, commerciallyavailable form of cellulose such as wood, pulp, cotton hemp, ramie andthe like. Preferably, the cellulose is a solid matrix of celluloseacetate or paper. More preferably, a thin sheet of paper is employed,and most preferably, Whatman 3 MM paper is employed for making the metalcharged IDA cellulose of the present invention.

Metal charged IDA cellulose of the present invention is charged with ametal salt. These metal salts are well recognized in the art.Preferably, the metal salts include metals which are cations that chargethe IDA cellulose. These metals include nickel, zinc, iron, cobalt,cadmium, manganese and magnesium. The most preferred metal to charge IDAcellulose is Ni²⁺. The structure of Ni²⁺-charged IDA cellulose is shownin FIG. 1, segment 3.

In one embodiment of the present invention, the metal charged IDAcellulose is utilized to detect a protein of interest, for example,proliferating cell nuclear antigen (PCNA). Preferably, the protein ofinterest has a polyhistidine region or a histidine tag (his-tag). Thehistidine tag can be naturally occurring or provided by a vector thatencodes the polyhistidine region. Typically, his-tagged proteins containa string of at least about four to at least about ten consecutivehistidine residues at either the amino (N)-terminal or carboxyl(C)-terminal end of the protein of interest. Most preferably, theprotein of interest has a histidine tag of at least about four to atleast about six histidine residues.

The metal charged IDA cellulose of the present invention may be preparedaccording to the reaction scheme shown in FIG. 1, with startingreactants known in the art. Preferably, the starting reactant iscellulose paper or cellulose acetate membrane and more preferably,Whatman 3 MM paper (shown in FIG. 1, segment 1). Preferably, the metalcharged IDA cellulose is prepared by wetting the cellulose or cellulosemembrane with a suitable liquid, for example, water. The wet celluloseis then contacted or reacted with an ether under basic conditions toform cellulose epoxide (epoxy-cellulose) . Most preferably, the etherused is 1,4-butanediol diglycidyl ether.

The reaction is carried out under basic conditions. As used herein,basic conditions arise upon contacting the wet cellulose with a suitablebasic solution, capable of dissociating OH⁻ ions in solution. MostPreferably, the basic conditions include a pH of about 12 or greater. Anexample of suitable basic solutions include combinations of NaBH₄,Na₂CO₃ and NaOH.

The cellulose-epoxide is reacted or contacted with a suitable organicacid to form iminodiacetic acid (IDA) cellulose. The reaction schemethat can be used to form IDA cellulose is shown in FIG. 1, segment 2. Anexample of a suitable organic acid is iminodiacetic acid. Mostpreferably, the concentration of iminodiacetic acid reacted with theepoxy cellulose is about 0.1M to about 0.2M.

Metal charging the iminodiacetic acid-cellulose may be accomplished byincubating the IDA cellulose with a suitable metal salt. Preferably, themetal salts include cationic metals capable of charging the IDAcellulose. These metals include nickel, zinc, iron, cobalt, cadmium,manganese and magnesium. Suitable metal salts include NiCl₂, CoCl₂,ZnCl₂, MgCl₂, and the like. Most preferably, the metal salt employed tocharge IDA cellulose of the present invention is NiCl₂. This metal saltdissociates into Ni²⁺ cations which charge the IDA cellulose. Mostpreferably, at least about 50 mM of NiCl₂ is used.

Preferably, IDA cellulose is incubated at about 25° C. with the metalsalt to produce the metal charged IDA cellulose. The incubation periodcan vary depending on the metal salt used. Preferably, the period ofincubation is at least about five minutes to at least about two hours,more preferably, at least about ten minutes to at least about one hour,and most preferably, for about ten minutes to about thirty minutes.

The amounts and concentrations of reactants used to make the metalcharged IDA cellulose can vary depending on the quantity of thecellulose contacted with the reactants and the amount of metal chargedIDA cellulose produced. In any event, the practitioner is guided byskill and knowledge in the field, and the present invention includeswithout limitation amounts of reactants which are effective to producethe metal charged IDA cellulose of the present invention.

The metal charged IDA cellulose of the present invention is useful forbinding any protein or any test sample having a histidine tag.Accordingly, the present invention provides a method for screening atest sample by contacting metal charged IDA cellulose with the testsample, washing the IDA cellulose and detecting the his-tagged portionof the sample that remains immobilized on the metal charged IDAcellulose.

Examples of test samples include any macromolecule, such as proteinsincluding isolated natural proteins, recombinantly produced or clonedproteins, a library of cloned proteins, synthetic proteins, peptides,amino acids, cell lysates, hormones, enzymes, and the like. Themacromolecule can be isolated from cell lysates before applying it tothe metal charged IDA cellulose. Preferably, the cell lysate is appliedto wet metal charged IDA paper overlaid with a filter. A suitable filterfor use with the present invention is cellulose acetate membrane.

The method of the present invention involves washing the metal chargedIDA cellulose to remove contaminants and other non-his-taggedmacromolecules from the metal charged IDA cellulose. Suitable agentsused to wash the metal charged IDA cellulose include combinations ofwater, Bis-Tris, Triton X-100, NaCl, low concentrations of imidazole(such as 10 mM), glycerol and BSA (bovine serum albumin).

Metal charged IDA cellulose of the present invention immobilizes orbinds macromolecules, such as proteins having histidine tags. Theseproteins can easily be detected or measured by various means. Forexample, the immobilized protein can be detected using an antibody thatbinds to or associates with particular region(s) of the immobilizedprotein. Preferably, such antibodies are monospecific. An example of amonospecific antibody used to detect his-tagged human PCNA is mAb PC10(Oncogene Sciences, Uniondale, N.Y.). Alternately, the immobilizedprotein can be detected with a stain, for example, Coomassie blue oramino black.

Furthermore, once the test sample is immobilized on the metal chargedIDA cellulose, qualitative or quantitative information can be providedabout the test sample analyzed. For example, when the test samplederives from cloned prokaryotic or eukaryotic cells, the amount ofprotein expressed or produced by the particular clone can be quantifiedor measured. Measurement of the quantity of protein expressed can beaccomplished using methods known within the art. Preferably, suchmeasurement is conducted densitometrically to determine the proteinexpression level of the clone. Accordingly, the invention is highlyuseful for high throughput screening of a large number of mutants andcloned proteins for expression and functional activity.

Qualitatively, the functional activity of the his-tagged test sampleimmobilized on the metal charged IDA cellulose can be tested byfunctional assays using methods known by those skilled in the art. Inaddition, immunoassays and immunological techniques such as RIA andWestern blotting can be employed.

It has been discovered that some proteins having a polyhistidine region,such as PCNA, retain biological or functional activity even after beingimmobilized on the metal charged IDA cellulose. For example, his-taggedPCNA, once immobilized on the metal charged IDA cellulose, isfunctionally active based on its ability to stimulate DNA polymeraseactivity in situ of purified calf thymus pol δ. Accordingly, the presentinvention includes isolating his-tagged proteins in an active form wherethe isolated protein retains biological or functional activity. Examplesof biological or functional activity of the protein of interest includeenzyme activity or binding specificity for a particular binding site.

In another embodiment, the present invention provides a method fordetermining protein expression. This method includes introducing intocells a vector comprising a nucleic acid that encodes a protein ofinterest having a polyhistidine region. Using methodology well known inthe art, nucleic acid or recombinant DNA molecules can be constructed(cDNA library) or are readily commercially available. Similarly,numerous vectors, including eukaryotic and prokaryotic vectors arecommercially available to the artisan. Preferably, the vector carries alibrary of random point mutants with a modified nucleotide sequence thatexpress the his-tagged protein. For example, a suitable vector forhis-tagged PCNA is pQE30 which is commercially available from Qiagen,Valencia, Calif.

The vector may be introduced into the host cell by methods known in theart, such as transforming or transfecting the host cell with a livevirus. The host cells can be, for example, prokaryotic cells such asEscherichia coli, Staphylococcus aureus, or eukaryotic cells such as ayeast, e.g., Saccharomyces cerevisiae, or cultured mammalian cells frommulticellular organisms, e.g., Chinese hamster ovary cells (CHO) or Coscells. An example of a commercially available bacterial cell line thatis suitable for the present invention is E. coli, strain M15 fromQiagen.

Using methodology well known in the art, the transformed or transfectedcells are grown to express the his-tagged protein on a suitable mediasuch as in liquid culture, on plates or in wells. Most preferably, thetransformed or transfected cells are grown in colonies on plates.

The method of the present invention is performed by transferring theprotein of interest to a metal charged IDA cellulose. Preferably, thetransfer of the protein of interest is accomplished using methods knownin the art, such as using replica transfer from a membrane support orwith a pipet. Typically, the original transformed or transfectedcolonies are contacted with a membrane support, such as celluloseacetate, in which case about 10% of each colony is transferred to themembrane support. The resulting cells on the membrane support arereplicas or copies of the original colonies of transformed ortransfected cells. These replicas are contacted with the metal chargedIDA cellulose. Accordingly, a replica from each original colony istransferred to the metal charged IDA cellulose.

In an alternate embodiment, the transfected cells are lysed in situ torelease intracellular proteins including the protein of interest usinglysing agents well known in the art. Examples of lysing agents includelysozyme in combination with EDTA, SDS, and/or Triton X-100. Once thecells are lysed, the lysates or intracellular proteins are transferredto the metal charged IDA cellulose.

The method of the present invention includes denaturing a protein havinga polyhistidine region or a histidine tag. As used herein the term“denaturing” includes treating a protein with a denaturant that resultsin loss of the tertiary or native structure of the protein. Denaturingconditions lead to the loss of biological or functional activity of theprotein. Examples of commonly used denaturants include urea, guanidiniumchloride, guanidinium thiocyanate, and detergents such as SDS.

The method of the present invention involves contacting the denaturedprotein with metal charged IDA cellulose to transfer and bind theprotein to the cellulose under denaturing conditions. The denaturedprotein may be transferred to the metal charged IDA cellulose asdiscussed above.

Once the denatured protein is immobilized or bound to the metal chargedIDA cellulose, the protein is then renatured, preferably in situ or onthe metal charged IDA cellulose. As used herein, the term “renaturing”includes treating the protein with a denaturant that results in thereturn of the tertiary or native structure to the protein. Renaturingconditions lead to the restoration of biological activity of theprotein. Examples of commonly used renaturants include urea, guanidiniumthiocyanate, sodium chloride and water.

Preferably, the renaturing occurs by removing the denaturant, morepreferably, by washing or diluting the denaturant, and most preferably,by diluting the denaturant with water in situ or on the metal chargedIDA cellulose. In an alternate embodiment, the protein is renatured bywashing the protein bound to the metal charged IDA cellulose with adenaturant of decreasing concentration. For example, if the denaturantused is 8M of urea, then the denatured protein can be renatured bywashing the protein with a denaturant of decreasing concentration, forexample, 6M concentration of urea to about 2M concentration of urea,until the protein renatures. Preferably, functional assays can beperformed after each decreasing concentration of the denaturant isapplied to the protein.

In one embodiment of the present invention, the renatured protein isrecovered or eluted from the metal charged IDA cellulose usingconventional methods known in the art for eluting proteins from IDAresins. For example, the protein can be eluted by washing the resin withcombinations of water, Bis-Tris, Triton X-100, NaCl, high concentrationsof imidazole (such as 400 mM), glycerol and BSA (bovine serum albumin).

The metal charged IDA cellulose of the present invention can be placedin wells of a manifold such as plexiglass, wood, plastic and the like.Subsequently, the test sample is placed in each well and washed, andhis-tagged macromolecules are detected on the metal charged IDAcellulose. Accordingly, a large number of test samples can be screenedfor his-tags and the method is amenable to automation. Thus, themanipulation of each individual test sample is avoided.

The examples below are directed to the metal charged IDA cellulose ofthe present invention. Further examples demonstrate methods for usingthe metal charged IDA cellulose as a two-dimensional matrix to screenreplicas of bacterial cultures or colonies after induction and celllysis in situ for his-tagged proteins.

More particularly, using his-tagged but otherwise wildtype PCNA, an insitu assay is provided that is dependent on the ability of his-taggedPCNA to stimulate added pol δ with an exogenous template-primer. Thisassay was used together with random mutagenesis to identify a PCNApoint-mutation that stimulates enhanced pol δ-catalyzed DNA synthesisbeyond a model abasic template site. Accordingly, one of ordinary skillin the art, given the present disclosure, will understand that similarapproaches may be taken to study other proteins or macromolecules thathave a his-tag.

EXAMPLES

Examples have been set forth below for purpose of illustration and todescribe the best mode of the invention at the present time. The scopeof the invention is not to be in any way limited by the examples setforth herein.

Example 1

Materials and Methods

Much of the methodology was described (Refs. 1, 7, 8, 12-14). SDS-PAGEwas according to Laemmli (Ref. 15) as modified by Fisher et al., (Ref.16) on minigels. For immunoblots, proteins were transferredelectrophoretically to nitrocellulose (Ref. 17) and resulting replicaswere probed with antibodies. Reactivity was visualized colorimetrically(Ref. 18) with phosphatase-conjugated goat anti-IgG antibodies (Refs.19, 20) and a one-solution phosphatase substrate (Kirkegaard and Perry,Gaithersburg, Md.). Immunologic detection of human PCNA was with mousemonoclonal antibody (mAb) PC10 (Oncogene Sciences, Uniondale, N.Y.).Detection of Drosophila PCNA was with affinity purified polyclonalrabbit anti-Drosophila PCNA antibodies (Ref. 12). Restrictionendonucleases were from Boehringer (Indianapolis, Ind.) and were usedaccording to the vendor's instructions. Acrylamide and methylenebis-acrylamide were from Eastman (Rochester, N.Y.) and were furtherpurified by adsorption to an ion exchange resin. DNA sequencingperformed in both directions was according to Sanger et al. (Ref. 21)using a fluorescence-based method and an ABI 373 (Applied Biosystems,Foster City, Calif.) automated DNA sequencer.

Proteins. PCNA was purified to apparent homogeneity from calf thymus(Ref. 2) as was pol δ (Refs. 14, 22). Human PCNA cDNA was cloned into abacterial expression vector and purified from an E. coli extract (Ref.23). D. melanogaster PCNA was purified to apparent homogeneityidentically after bacterial expression (Ref. 24). Where indicated,purification of his-tagged proteins included chromatography onNi²⁺-IDA-Sepharose (Pharmacia). A his-tag was added to the NH₂-terminiof both human and Drosophila PCNA by cDNA insertion into pQE30 (Qiagen,Valencia, Calif.) using BamH1 and HindIII restriction sites.

Nucleic Acids. Templates and primers of defined sequence weresynthesized conventionally. Before use, all were purified by standarddenaturing PAGE (Ref. 25). All other DNA manipulations were according tostandard techniques (Ref. 25).

Example 2

Preparation of Ni²⁺-charged IDA-cellulose

The procedure used to derivatize Whatman 3 MM paper is shown in FIG. 1.Before reaction, the paper was washed with H₂O. It was then reacted for6.5 h at 25° C. with 4% (v/v) 1,4-butanediol diglycidyl ether in thepresence of 0.4 N NaOH and 12 mM NaBH₄. Afterward, the resultingcellulose-epoxide was washed with H₂O and reacted for 16 h at 55° C.with 0.15 M IDA in the presence of 0.8 M Na₂CO₃ (unbuffered) and 12 mMNaBH₄. Afterward, the resulting IDA-cellulose was washed with H₂O and ifdesired, stored wet in 100 mM Tris-HCl pH 8 for up to 6 months at 4° C.Immediately before use, IDA-cellulose (washed again with H₂O if stored)was charged with Ni²⁺ by incubation for 20 min at 25° C. with 50 mMNiCl₂. After charging, Ni²⁺-IDA-paper was washed, first with water andthen with 40 mM Bis-Tris pH 6.8, 0.01% (v/v) Triton X-100, 0.7 M NaCl,0.1% (w/v) BSA and 10% (v/v) glycerol. By incubation with other salts(e.g., CoCl₂), charging with other metals can be accomplished.

In summary, FIG. 1 illustrates the chemical treatment of cellulose toimmobilize his-tagged proteins. The complete treatment of eitherregenerated cellulose membrane or Whatman 3 MM paper to produceNi²⁺-charged IDA-cellulose is shown in segments 1-3.

Example 3

Bacterial Growth, Induction and Transfer of Proteins to Ni²⁺-chargedIDA-paper

E. coli, strain M15 (Qiagen), were transformed with pQE30 with orwithout the gene encoding PCNA under control ofisopropyl-β-D-thiogalactoside (IPTG). Cells were grown either ascolonies on cellulose acetate membranes (0.45 μm pore size, MSI,Westboro, Mass.) laid over standard 1.5% agar plates in LB medium or inliquid culture, also in LB medium. Unmodified human PCNA under IPTGcontrol in pT7 (Ref. 23) was maintained and induced in bacterial strainBL21(DE3). Unmodified Drosophila PCNA, also under IPTG control but inpTDT7 (Ref. 24) was maintained and induced similarly.

E. coli, strain M15 (Qiagen), were transformed with pQE30 with orwithout the gene encoding PCNA under control ofisopropyl-β-D-thiogalactoside (IPTG). Cells were grown either ascolonies on cellulose acetate membranes (0.45 μm pore size, MSI,Westboro, Mass.) laid over standard 1.5% agar plates in LB medium or inliquid culture, also in LB medium. Unmodified human PCNA under IPTGcontrol in pT7 (Ref 23) was maintained and induced in bacterial strainBL21(DE3). Unmodified Drosophila PCNA, also under IPTG control but inpTDT7 (Ref. 24) was maintained and induced similarly.

Macromolecular transfer was performed after induction and cell lysis.For colonies, colony-containing membranes were replicated, transferredto standard agar plates containing in addition, 1 mM IPTG and inducedovernight at 37° C. Induction of liquid cultures was performed by addingIPTG to a final concentration of 1 mM and continued incubation for 3 hat 37° C. Before transfer, the Ni²⁺-IDA-paper was washed with lysisbuffer and then kept wet with this solution. Cells were lysed byaddition of a solution of 1 mg/ml of hen egg white lysozyme in “lysisbuffer” (50 mM Tris-HCl pH 8, 0.1 M NaCl, 0.1% [w/v] BSA, 0.1% [v/v]Triton X-100 and 5% [v/v] glycerol); at the same time, macromoleculeswere transferred passively for 1 h (Ref. 16) from the cellulose acetatemembrane on which colonies were grown through a similar membrane (0.45μm pore size) which acted as an inert filter to a sheet ofNi²⁺-IDA-paper placed on top of the second cellulose acetate membrane.Lysis buffer was used for transfer. Alternatively, lysate was preparedfrom induced cells according to standard protocols (Ref. 25) and applieddirectly to wet Ni²⁺-IDA-paper overlaid with a cellulose acetatemembrane which acted as an inert filter (i.e., lysate was applied to theNi²⁺-IDA-paper through a membrane by direct application to themembrane). After transfer, the Ni²⁺-IDA-paper was washed four times, 30min each time, each in at least 2 ml/cm² of paper. The first two washeswere in 40 mM Bis-Tris pH 6.8, 0.01% (v/v) Triton X-100, 0.7 M NaCl,0.1% (w/v) BSA, 10 mM imidazole and 10% (v/v) glycerol. The second twowashes were in the same solution lacking NaCl and imidazole.

Example 4

In situ Detection of DNA Polymerase δ

Solution assays of pol δ were performed according to Ng et al. (Ref.14). In situ assays were performed in a final volume of 30 μl/cm² ofpaper. Before incubation, sheets of protein-containing Ni²⁺-IDA-paperwere “sandwiched” between two sheets of hydrated but otherwiseunmodified 3 MM paper (hydrated with solution containing 40 mM Bis-TrispH 6.8, 6 MM MgCl₂, 0.1% BSA and 10% glycerol). Sheets of wetN²⁺-IDA-paper prepared exactly as described above and to whichmacromolecules had been transferred, were placed in empty bacterialculture plates or plastic bags along with purified pol δ (0.1 μg/ml),dATP, dTTP and dCTP, each at 10 μM, 50 μCi/ml of [α-³²P]dGTP, 300 nM21-mer DNA primer annealed to a stoichiometrically equal amount of30-mer DNA template, 20 mM imidazole, 40 mM Bis-Tris pH 6.8, 6 mM MgCl₂,0.1% BSA and 10% glycerol. Incubations followed and were at 37° C. for180 min. Afterwards, reacted Ni²⁺-IDA-paper sheets were washedextensively in solution containing 5% (w/v) trichloroacetic acid andfinally subjected to analysis with a Molecular Dynamics 445 SIPhosphorImager.

Example 5

Random Mutagenesis of His-tagged Human PCNA

Random mutagenesis of human PCNA cDNA in pT7 was by polymerase chainreaction (PCR) according to Zhou et al. (Ref. 26). The primers used were5′-AGT TAG GAT CCA TGT TCG AGG CGC GC (SEQ ID NO:1) and 5′-TCT ACA AGCTTA AGA TCC TTC TTC ATC C (SEQ ID NO:2), complementary to PCNA sequencesat either end respectively, of the PCNA insert. PCR were performed inmultiple 100-μl reactions for 30 cycles, each with the followingtemperature profile: 1.2 min at 94° C.; 1.2 min at 50° C.; 2.5 min at72° C. About 20% of the clones contained PCNA amino acid mutations(determined empirically). After PCR, the DNA product was purified byphenol/chloroform extraction and ethanol precipitation, treatedexhaustively with HindIII and BamH1, and finally purified by agarose gelelectrophoresis. The restricted, purified fragment was ligated intopQE30 which had first been similarly restricted and the ligation mixturewas used to transform M15 cells.

Example 6

Ni²⁺-IDA-paper Binds His-tagged but not Unmodified PCNA

Both human and Drosophila PCNA were modified to contain NH₂-terminalhis-tags, partially purified and compared with wild-type PCNA, similarlypurified from bacterial extracts. As expected, his-tagged PCNA migratedslightly more slowly than the respective wild-type homologs duringSDS-PAGE (FIG. 2A). Immunoblot analyses (FIGS. 2B and 2C) revealed thatin neither case did introduction of the his-tag affect reactivity withspecific antibodies.

More specifically, FIG. 2 shows the results of bacterial expression andpurification of unmodified and NH₂-terminally his-tagged PCNA; SDS-PAGEand immunoblot analysis performed. Only regions of interest are shown(FIGS. 2A-C). Referring to FIG. 2A, the gel was stained with Coomassieblue. Immunoblot analysis (shown in FIGS. 2B and 2C) of partiallypurified PCNA from a gel loaded and run in parallel with the one shownin FIG. 2A. Immunoblot probed with mAb PC10 is shown in FIG. 2B.Immunoblot probed with affinity purified rabbit polyclonalanti-Drosophila PCNA antibody is shown in FIG. 2C. In all panels, lanes1 were loaded with unmodified human PCNA; lanes 2 were loaded withhis-tagged human PCNA; lanes 3 were loaded with unmodified DrosophilaPCNA; and lanes 4 were loaded with his-tagged Drosophila PCNA.Immunoblot analysis revealed that the introduction of the his-tag didnot affect reactivity with specific antibodies.

To test Ni²⁺-IDA-paper, we expressed PCNA both with and withoutNH₂-terminal his-tags. Monoclonal antibodies were used to evaluate humanPCNA binding after various treatments of the matrix (FIG. 3). In allcases, bacterial lysates containing either his-tagged (FIG. 3 spotslabeled 1) or unmodified (FIG. 3 spots labeled 2) human PCNA, wereapplied directly to the two-dimensional matrix. Afterward, matrixsegments were subjected to immunoblot-type analyses with mAb PC10. WhenNi²⁺-IDA-paper was used, only his-tagged PCNA could be detected (FIG.3A). When IDA-paper was not charged with metal (i.e., not incubated withNiCl₂), little or no PCNA was detected (FIG. 3B). Similarly negativeresults were seen when unmodified 3 MM paper was used instead ofIDA-paper (FIG. 3C) and when Ni²⁺-charged IDA-paper was incubated for 5min in 600 mM imidazole after lysate application (FIG. 3D). In contrast,binding of PCNA was readily detected when lysates were applied tonitrocellulose (FIG. 3E). PCNA binding to nitrocellulose however, wasindependent of the NH₂-terminal his-tag.

In summary, FIG. 3 illustrates binding of his-tagged and unmodified PCNAto various supports. Bacterial lysates containing equal concentrations(calibrated by SDS-PAGE and immunoblot analysis) of either his-taggedhuman PCNA (spots labeled 1) or unmodified human PCNA (spots labeled 2)were applied as follows: FIG. 3A, to Ni²⁺-charged IDA-paper; FIG. 3B, touncharged IDA-paper; FIG. 3C, to unmodified 3 MM paper that had firstbeen incubated with 50 mM NiCl₂ as specified in FIG. 1; FIG. 3D, toNi²⁺-IDA-paper but after lysate application, it was incubated for 5 minin 600 mM imidazole; and FIG. 3E, to nitrocellulose. All were thensubjected to standard immunoblot-type analysis; mAb PC10 was used as theprimary and phosphatase-conjugated goat anti-mouse IgG was used as thesecondary antibody. Identical results were obtained with his-taggedDrosophila PCNA versus unmodified Drosophila PCNA (not shown).

Example 6

His-tagged Human PCNA Bound to Ni²⁺-IDA-paper can Stimulate Calf ThymusDNA Polymerase δ

Ni²⁺-IDA-paper was prepared and bacterial lysates containing eitherhistagged (FIGS. 4A-C, spots labeled 1) or unmodified (FIGS. 4A-C, spotslabeled 2) human PCNA were applied. After washing, segments wereincubated with: FIG. 4A, complete DNA polymerase incubation mixturesupplemented with purified calf thymus pol δ and the exogenous DNAtemplate-primer shown in FIG. 4D; FIG. 4B, incomplete DNA polymeraseincubation mixture supplemented with exogenous template-primer butlacking pol δ; or FIG. 4C, incomplete DNA polymerase incubation mixturesupplemented with purified pol δ but lacking exogenous template-primer.Intense reactivity was observed, only when his-tagged, as opposed tounmodified PCNA was present in the bacterial lysate (FIG. 4A, comparespot 1 versus spot 2), and was dependent on exogenous polymerase (FIG.4, compare panel A with panel B) as well as exogenous DNA substrate(FIG. 4, compare panel A with panel C).

Recombinant human PCNA expressed in E. coli and authentic calf thymusPCNA can be used interchangeably (Refs. 8, 12, 23). In contrast,Drosophila PCNA, although 73% identical to human PCNA in primarysequence (Ref. 24) and able to substitute qualitatively for human PCNAin some contexts (Ref. 12), could only stimulate calf thymus pol δweakly (<10% as well) (Ref. 13). Bacterial lysates containing his-taggedhuman PCNA, his-tagged Drosophila PCNA or lacking PCNA were applied toNi²⁺-IDA-paper. The paper was then washed and incubated in completereaction mix including purified calf thymus pol δ and DNAtemplate-primer (30-21-mer; see FIG. 4D). Intense reactivity wasobserved corresponding to the spot of human PCNA (FIG. 4E spot 1),whereas almost no reactivity was observed corresponding to the spot ofDrosophila PCNA (FIG. 4E spot 2) and no reactivity was observed in theregion lacking PCNA (FIG. 4E spot 3). The presence of Drosophila PCNAwhere applied was confirmed by standard immunoblot-type analysis withaffinity purified rabbit anti-Drosophila PCNA antibodies (not shown butsee FIGS. 2C and 3A).

In summary, FIG. 4 shows in situ reactivity of his-tagged human PCNA,purified calf thymus pol δ and exogenous DNA template-primer.Ni²⁺-IDA-paper was prepared and washed, bacterial lysate was applied andfurther washing was performed, exactly as in FIG. 3. Referring to FIGS.4A-C, lysates applied to spots labeled 1 contained his-tagged human PCNAand lysate applied to spots labeled 2 contained unmodified human PCNA.The segment shown in FIG. 4A was incubated for 180 min at 37° C. incomplete polymerase reaction mix containing both purified pol δ andexogenous DNA template-primer of: 5′-GAA TTC AAG CTT GTC GAC AGA-3′ (SEQID NO: 3) and 3′-CTT AAG TTC GAA CAG CTG TCT AGA GAC GTC-5′ (SEQ ID NO:4) as shown in FIG. 4D. These nucleotide sequences were chemicallysynthesized using methods known in the art (Ref. 30). The segment inFIG. 4B was incubated identically in incomplete mix lacking exogenouspol δ. The segment in FIG. 4C was incubated identically in incompletemix lacking exogenous DNA template-primer. After incubation, allsegments were washed extensively in 5% (w/v) trichloroacetic acid andsubjected to PhosphorImager analysis. Referring to FIG. 4E, in situreactivity of purified calf thymus pol δ and exogenous DNAtemplate-primer requires his-tagged human PCNA and is not seen withhis-tagged Drosophila PCNA or in the absence of PCNA. Ni²⁺-IDA-paper wasprepared and washed, bacterial lysate was applied and further washingwas performed, exactly as in FIG. 3. The lysate applied to spot 1contained his-tagged human PCNA. The lysate applied to spot 2 containedan equal amount of his-tagged Drosophila PCNA (calibrated by SDS-PAGEand immunoblot analysis). The lysate applied to spot 3 lacked his-taggedPCNA. After lysate application and washing, the segment shown wasincubated for 180 min at 37° C. in complete mix containing both purifiedpol δ and exogenous DNA template-primer, washed and subjected toPhosphorImager analysis, exactly as described above.

Example 7

Screening Colonies for Expression of Human PCNA Containing anNH₂-terminal His-tag

Colonies were grown on a 0.45 μm pore size cellulose acetate membrane(FIG. 5A). All cells were transformed with IPTG-inducible plasmids butonly half could express his-tagged human PCNA. The remainder containedthe same plasmid but without PCNA insert. Multiple replicas wereprepared. Expression was induced, colonies were lysed and proteins weretransferred passively to Ni²⁺-IDA-paper (Ref. 16). PCNA binding wasmonitored by immunoblot-type assay using mAb PC10 (FIG. 5B). In situassay with incubation mix lacking exogenous pol δ but containing DNAtemplate-primer (30-21-mer; see FIG. 4D) and [α-³²P]dGTP was negative(FIG. 5C). Negative results were similarly obtained when exogenoustemplate-primer (DNA) was omitted (i.e., [α-³²P]dGTP and exogenous pol δwere both included; not shown). Hence, neither bacterial DNApolymerases, dNTP binding proteins nor DNA were adsorbed to the paper,or if present, do not produce a measurable signal in this assay. Incontrast, positive results were obtained when the incubation mixcontained both purified calf thymus pol δ and an exogenoustemplate-primer. They were dependent on the presence of his-tagged humanPCNA (FIG. 5D). Non-specific protein staining (Coomassie blue) failed todetect any bacterial proteins bound to the Ni²⁺-IDA-paper after colonylysis and protein transfer (FIG. 5E).

In summary, FIG. 5 shows growth of bacterial colonies, specificimmobilization of his-tagged human PCNA on Ni²⁺-IDA-paper and in situreactivity of purified calf thymus pol δ. Bacterial colonies were grownon 0.45 μm pore size cellulose acetate membranes. Multiple replicas wereprepared. After induction and lysis, macromolecules were transferred toNi²⁺-IDA-paper. FIG. 5A shows bacterial colonies on a cellulose acetatemembrane. FIG. 5B shows Ni²⁺-IDA-paper after macromolecular transfersubjected to immunoblot-type analysis with mAb PC10. FIG. 5C showsNi²⁺-IDA-paper after macromolecular transfer subjected to DNA polymeraseanalysis with mix lacking exogenous pol δ, as in FIG. 4B. FIG. 5D showsNi²⁺-IDA-paper after macromolecular transfer subjected to DNA polymeraseanalysis with complete mix, as in FIG. 4A. FIG. 5E shows Ni²⁺-IDA-paperafter transfer and washing subjected to standard Coomassie bluestaining/destaining.

Example 8

Detectable Binding to Ni²⁺-IDA-paper Depends on Expression of aHis-tagged Protein

Because of the small amounts of material transferred and the relativeinsensitivity of protein staining, colonies expressing his-tagged PCNAcould not be distinguished from colonies not expressing any his-taggedprotein (FIG. 5E). However, much more material could be delivered bydirect application of lysates. Lysates from bacteria expressing eitherhis-tagged or unmodified human PCNA were applied to Ni²⁺-IDA-paper.Segments were washed, stained with Coomassie blue and destained. Intensestaining was seen on the segment to which lysate containing histaggedhuman PCNA was applied (FIG. 6A, panel 1). No staining was observed onthe segment to which lysate containing unmodified human PCNA was applied(FIG. 6A, panel 2). Similarly, no staining was observed when solutionscontaining 100 mg/ml of standard proteins (either bovine serum albuminor cytochrome C, both lacking his-tags) were applied to Ni²⁺-IDA-paper(not shown).

Scanning densitometry revealed that the intensity of Coomassie bluestaining observed was linearly dependent on the amount of lysatecontaining a his-tagged protein applied to Ni²⁺-IDA-paper up to a valueof about 59 μg/cm² of paper (9.4 μg of singly his-tagged human PCNAapplied to 0.16 square centimeters of Ni²⁺-IDA-paper; FIG. 6B). Althoughconsiderably more singly his-tagged protein could be bound, somedeviation from linearity was seen at higher values (FIG. 6B). Ourtechnique therefore promises to be useful for quantifying his-taggedprotein expression levels among otherwise similar transformants (ortransfectants).

In summary, FIG. 6 shows Coomassie blue staining of his-tagged human butnot unmodified human PCNA immobilized on Ni²⁺-IDA-paper; bothqualitative and quantitative analyses. FIG. 6A shows Coomassie bluestaining of bacterial lysates applied directly to Ni²⁺-IDA-paper. Thelysate applied in panel 1 contained his-tagged human PCNA. The lysateapplied in panel 2 contained an equal amount of unmodified human PCNA(calibrated by SDS-PAGE and immunoblot analysis). After washing, thesegments shown were subjected to standard Coomassie bluestaining/destaining. FIG. 6B shows densitometric quantification ofhis-tagged protein binding to Ni²⁺-IDA-paper. Singly-his-tagged humanPCNA was expressed in bacteria, purified to apparent homogeneity andapplied in varying quantities using a plexiglas manifold to 0.16 squarecentimeters of Ni²⁺-IDA-paper. Staining with Coomassie blue anddestaining were exactly as in FIG. 6A after which, bound protein wasquantified densitometrically. Plotted is the amount of protein applied(abscissa) versus the intensity of Coomassie blue staining (ordinate).

Example 9

The E⁸⁵>K PCNA Mutant; a Recombinant Molecule Identified by ScreeningNi²⁺-IDA-paper to which Randomly Mutagenized His-tagged PCNA was Bound

To screen for mutants, the cDNA coding for PCNA was randomly mutagenizedand inserted into pQE30 so as to introduce an NH₂-terminal his-tag.Lysates containing expressed proteins were applied to Ni²⁺-IDA-paper andthen screened using purified pol δ and synthetic template-primercontaining a model abasic template site (FIG. 7D). To control forvarying expression, a replicate filter was subjected to immunoblot-typeassay with mAb PC10 (FIG. 7A). Among the first group of 96, one mutantwas reproducibly detected (FIG. 7B); the mutant construct was designatedpQE-mutPCNA34.

Cells harboring pQE-mutPCNA34 were grown in liquid culture, induced andmutant PCNA purified free of contaminating nuclease(s) andpolymerase(s). Purification was tremendously facilitated by the his-tag.For all his-tagged proteins (including wild-type and E⁸⁵>K human PCNA;see FIG. 7C), purification included chromatography on Ni²⁺-IDA-Sepharose(Pharmacia). SDS-PAGE electropherograms of purified wild-type PCNA, bothwithout (FIG. 7C lane 1) and with (FIG. 7C lane 2) NH₂-terminal his-tagare shown. As expected, his-tagged PCNA moves perceptibly slower thanthe untagged protein. Also shown is purified protein encoded bypQE-mutPCNA34 (FIG. 7C lane 3). It too has a distinctive mobility,consistent with a single amino acid change. DNA sequencing performed inboth directions indicated that the mutation is E⁸⁵>K.

We compared NH₂-terminally his-tagged E⁸⁵>K mutant PCNA with wild typePCNA, both with and without NH₂-terminal his-tags. All stimulatedpurified pol δ identically on poly(dA)-oligo(dT) (not shown) butcompared with wild-type, E⁸⁵>K mutant PCNA stimulated extended DNAsynthesis by pol δ beyond model abasic template lesions (the originalbasis for screening) by about 40% as determined by PhosphorImager (FIG.7E). Based on the crystal structure of the PCNA trimer (Ref. 27), theE⁸⁵>K mutation is on the PCNA surface thought to contact DNA (FIG. 7F),and to face away from pol δ (Ref. 28).

In summary, FIG. 7 shows the results of screening for PCNA mutants thatpromote extended DNA synthesis past a model abasic template site. FIG.7A, shows Ni-IDA paper screened by immunoblot-type assay with mAb PC10.In FIG. 7B, paper was screened by in situ polymerase assay using as theonly added DNA, the template-primer of: 5′-GAA TTC AAG CTT GTC GACAGA-3′ (SEQ ID NO: 3) and 3′-CTT AAG TTC GAA CAG CTG TCT XGA GAC GTC-5′(SEQ ID NO:5), shown in FIG. 7D containing a model abasic residue at theposition designated X. The arrow denotes the single positive lysate fromculture number 34 detected reproducibly. FIG. 7C showsSDS-PAGE/Coomassie blue analysis of highly purified (apparentlyhomogeneous) recombinant PCNA used for all subsequent experiments; lane1, unmodified wild-type human PCNA; lane 2, NH₂-terminally his-taggedwild-type human PCNA; lane 3, NH₂-terminally his-tagged E⁸⁵>K mutanthuman PCNA. Standard proteins were subjected to SDS-PAGE simultaneously;their migration positions are indicated to the right of FIG. 7C. FIG. 7Dis lesion-containing template-primer used for the screen having thenucleotide sequence of 5′-GAA TTC AAG CTT GTC GAC AGA-3′ (SEQ ID NO: 3)and 3′-CTT AAG TTC GAA CAG CTG TCT XGA GAC GTC-5′ (SEQ ID NO: 5)containing a model abasic residue at the position designated X. As usedin the nucleotide sequence, X denotes a chemically modified sugarphosphate or a modified nucleotide without the pyrimidine or purinebase. These nucleotides and sugar phosphates were chemically synthesizedusing procedures of Takeshita, et al., J. Biol. Chem. 262, (10) 171-181(1987), the entire disclosure of which is incorporated herein byreference.

Shown in FIG. 7E are solution assays of pol δ in the presence andabsence of different apparently homogeneous PCNA molecules. Allreactions contained purified pol δ, dATP, dCTP, dTTP and [α-³²P]dGTP.Incubations were for 30 min at 37° C. Electrophoresis after incubationwas by standard denaturing PAGE on a 16% polyacrylamide gel. The gel wassubjected to routine PhosphorImager analysis. Incubations contained:lane 1, no PCNA; lane 2, unmodified wild-type human PCNA; lane 3,NH₂-terminally his-tagged wild-type human PCNA; lane 4, NH₂-terminallyhis-tagged E⁸⁵>K mutant human PCNA. Migration positions of the 30-mertemplate, the 21-mer primer and the 22-mer formed by incorporationopposite the model abasic site are shown as indicated to the right ofFIG. 7E. Note that the apparent difference between the pattern seen inlanes 1 and 2 and that previously published (Ref. 8) is due to the factthat 5′-³²P-labeled 21-mer primers and unlabeled deoxyribonucleotidetriphosphates were used previously, whereas present studies wereperformed entirely with unlabeled 21-mer primers and [α-³²P]dGTP. FIG.7F shows location of the point mutation found (E⁸⁵>K) superimposed onthe crystal structure of the PCNA trimer. The face shown is that thoughtto be directed away from pol δ during DNA synthesis.

The above examples clearly show the detection of protein binding tometal charged IDA paper by introducing a his-tag into that protein usinggenetic engineering. In addition, the present invention detectshis-tagged human PCNA functionally, by its ability to stimulate the DNApolymerase activity in situ of purified calf thymus pol δ. The method ofthe present invention, therefore allows immediate investigation bysystematic mutagenesis, of those PCNA residues important for stimulationof pol δ.

The metal charged IDA cellulose and methods of the present invention canbe used for several novel applications unrelated to PCNA and/or pol δ.As discussed above, we used two-dimensional his-tagged proteinimmobilization and the in situ assay of pol δ to identify PCNA mutationsthat enhance the ability of purified calf thymus polymerase δ toreplicate beyond a model abasic site. Thus far, we have found severalPCNA mutants that apparently promote enhanced synthesis beyond a modelabasic template site by mammalian pol δ, thereby demonstrating theutility of Ni²⁺-IDA paper.

REFERENCES

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Thus, while there have been described what are presently believed to bepreferred embodiments of the present invention, those skilled in the artwill realize that other and further modifications and changes can bemade without departing from the true spirit of the invention, and it isintended to include all such changes and modifications as come withinthe scope of the invention as pointed out in the claims appended hereto.

What is claimed is:
 1. A method for determining protein expression,comprising: (i) providing cells containing a vector comprising a nucleicacid encoding a protein of interest having a polyhistidine region, saidcells expressing the protein of interest; (ii) transferring the proteinof interest to metal charged iminodiacetic acid cellulose, immobilizingthe protein of interest on the metal charged iminodiacetic acidcellulose; and (iii) detecting the protein of interest immobilized onthe metal charged iminodiacetic acid cellulose.
 2. The method accordingto claim 1, wherein the metal charged iminodiacetic acid cellulose ismetal charged iminodiacetic acid cellulose paper.
 3. The methodaccording to claim 2, wherein the cells expressing the protein ofinterest are replicated onto a membrane support prior to transferring tothe metal charged iminodiacetic acid cellulose paper.
 4. The methodaccording to claim 2, wherein the method further comprises lysing thecells to release the protein of interest before transferring the proteinof interest to the metal charged iminodiacetic acid cellulose paper. 5.The method according to claim 2, wherein the protein of interestimmobilized on the metal charged iminodiacetic acid cellulose paper iswashed prior to detecting the protein of interest.
 6. The methodaccording to claim 1, wherein the polyhistidine region of the protein ofinterest is a histidine tag at the N-terminal end or at the C-terminalend.
 7. The method according to claim 1, wherein the protein of interestis proliferating cell nuclear antigen (PCNA).
 8. The method according toclaim 1, wherein the nucleic acid vector encodes a library of randompoint mutations in the protein of interest.
 9. The method according toclaim 1, wherein the nucleic acid that encodes the protein of interestis from a cDNA library.
 10. The method according to claim 1, wherein theprotein of interest immobilized on the metal charged iminodiacetic acidcellulose is quantified.
 11. The method according to claim 1, whereinthe protein of interest immobilized on the metal charged iminodiaceticacid cellulose is quantified densitometrically.
 12. The method of claim1, wherein the metal is selected from the group consisting of nickel,zinc, iron, cobalt, cadmium, manganese and magnesium.
 13. The methodaccording to claim 1, wherein the cells containing the vector areeukaryotic cells or bacterial cells.
 14. The method according to claim13, wherein the eukaryotic cells are mammalian cells.
 15. The methodaccording to claim 14, wherein the mammalian cells are COS cells or CHOcells.
 16. The method according to claim 14, wherein the mammalian cellsare human cells.
 17. The method according to claim 13, wherein theeukaryotic cells are yeast cells.
 18. The method according to claim 13wherein the bacterial cells are E. coli cells or S. aureus cells. 19.The method according to claim 2, wherein the following steps areinserted between steps (i) and (ii): (a) preparing a replica of thecells that have the vector on a membrane support; and (b) lysing thereplica on the membrane support in situ to release the protein ofinterest.
 20. The method according to claim 19, further comprising astep of expressing the protein of interest in the replica, between step(a) and step (b).
 21. The method according to claim 19, wherein theprotein of interest immobilized on the metal charged iminoacetic acidpaper in step (ii) is washed prior to detecting the protein of interestimmobilized on the metal charged iminoacetic acid paper in step (iii).22. The method according to claim 19, wherein the membrane support iscellulose acetate.
 23. The method according to claim 19, wherein theprotein of interest immobilized on the metal charged iminodiacetic acidcellulose is quantified.
 24. The method according to claim 19, whereinthe protein of interest immobilized on the metal charged iminodiaceticacid cellulose is quantified densitometrically.
 25. The method accordingto claim 19, wherein the polyhistidine region of the protein of interestis a histidine tag at the N-terminal end or at the C-terminal end. 26.The method according to claim 19, wherein the protein of interest isproliferating cell nuclear antigen (PCNA).
 27. The method according toclaim 26, wherein the PCNA is human PCNA or D. melanogaster PCNA. 28.The method according to claim 19, wherein the nucleic acid vectorencodes a library of random point mutations in the protein of interest.29. The method according to claim 19, wherein the nucleic acid thatencodes the protein of interest is from a cDNA library.
 30. A method fordetermining a functional activity of a protein of interest, comprising:(i) providing cells containing a vector comprising a nucleic acidencoding a protein of interest having a polyhistidine region, said cellsexpressing the protein of interest; (ii) transferring the protein ofinterest to metal charged iminodiacetic acid cellulose, immobilizing theprotein of interest on the metal charged iminodiacetic acid cellulose;and (iii) determining the functional activity the protein of interestimmobilized on the metal charged iminodiacetic acid cellulose.
 31. Themethod according to claim 30, wherein the metal charged iminodiaceticacid cellulose is metal charged iminodiacetic acid cellulose paper. 32.The method according to claim 31, wherein the cells expressing theprotein of interest are replicated onto a membrane support prior totransferring to the metal charged iminodiacetic acid cellulose paper.33. The method according to claim 31, wherein the method furthercomprises lysing the cells to release the protein of interest beforetransferring the protein of interest to the metal charged iminodiaceticacid cellulose paper.
 34. The method according to claim 30, wherein theprotein of interest immobilized on the metal charged iminodiacetic acidcellulose paper is washed prior to determining the functional activityof the protein of interest.
 35. The method according to claim 30,wherein the polyhistidine region of the protein of interest is ahistidine tag at the N-terminal end or at the C-terminal end.
 36. Themethod according to claim 30, wherein the protein of interest isproliferating cell nuclear antigen (PCNA).
 37. The method according toclaim 30, wherein the nucleic acid vector encodes a library of randompoint mutations in the protein of interest.
 38. The method according toclaim 30, wherein the nucleic acid that encodes the protein of interestis from a cDNA library.
 39. The method according to claim 30, whereinthe functional activity of the protein of interest immobilized on themetal charged iminodiacetic acid cellulose is quantified.
 40. The methodaccording to claim 30, wherein the functional activity of the protein ofinterest is binding specificity, enzyme activity or stimulation of anenzyme activity.
 41. The method according to claim 30, wherein thefunctional activity of the detected protein of interest is stimulationof δ-polymerase activity.
 42. The method according to claim 30, whereinthe metal is selected from the group consisting of nickel, zinc, iron,cobalt, cadmium, manganese and magnesium.
 43. The method according toclaim 30, wherein the cells containing the vector are eukaryotic cellsor bacterial cells.
 44. The method according to claim 43, wherein theeukaryotic cells are mammalian cells.
 45. The method according to claim44, wherein the mammalian cells are COS cells or CHO cells.
 46. Themethod according to claim 44, wherein the mammalian cells are humancells.
 47. The method according to claim 43, wherein the eukaryoticcells are yeast cells.
 48. The method according to claim 43 wherein thebacterial cells are E. coli cells or S. aureus cells.
 49. The methodaccording to claim 31, wherein the following steps are inserted betweensteps (i) and (ii): (c) preparing a replica of the cells that have thevector on a membrane support; and (d) lysing the replica on the membranesupport in situ to release the protein of interest.
 50. The methodaccording to claim 49, further comprising a step of expressing theprotein of interest in the replica, between step (a) and step (b). 51.The method according to claim 50, wherein the protein of interestimmobilized on the metal charged iminoacetic acid paper in step (ii) iswashed prior to detecting the protein of interest immobilized on themetal charged iminoacetic acid paper in step (iii).
 52. The methodaccording to claim 42, wherein the membrane support is celluloseacetate.
 53. The method according to claim 50, wherein the polyhistidineregion of the protein of interest is a histidine tag at the N-terminalend or at the C-terminal end.
 54. The method according to claim 50,wherein the protein of interest is proliferating cell nuclear antigen(PCNA).
 55. The method according to claim 54, wherein the PCNA is humanPCNA or D. melanogaster PCNA.
 56. The method according to claim 49,wherein the nucleic acid vector encodes a library of random pointmutations in the protein of interest.
 57. The method according to claim49, wherein the nucleic acid that encodes the protein of interest isfrom a cDNA library.
 58. The method according to claim 49, wherein thefunctional activity of the detected protein of interest is bindingspecificity, enzyme activity or stimulation of an enzyme activity. 59.The method according to claim 58, wherein the functional activity of thedetected protein of interest is stimulation of δ-polymerase activity.